In vitro assembly of a functional nucleocapsid from the negative-stranded genome RNA of a defective interfering particle of vesicular stomatitis virus

Abstract

The template for transcription and replication of negative-stranded RNA viruses is a ribonucleoprotein structure, the nucleocapsid. We have developed a system that supports assembly of the negative-stranded RNA genome of a defective interfering (DI) particle of vesicular stomatitis virus (VSV) into a nucleocapsid in vitro. This system uses extracts from wild-type VSV-infected cells as a source of proteins to encapsidate the RNA. In vitro assembled nucleocapsids were compared to in vivo-derived nucleocapsids by the following characteristics: nuclease resistance of the encapsidated RNA, CsCl density banding of labeled RNA in a position coincident with nucleocapsids, correct sedimentation rate in sucrose gradients, the presence of the nucleocapsid protein on the nucleocapsids, and the infectivity of the in vitro assembled nucleocapsids. We conclude that the system we present is capable of assembling the isolated genome of a rhabdovirus DI particle into nucleocapsids indistinguishable from those produced during the course of intracellular DI replication.